The liver, an organ which is present in vertebrates and other animals, plays a major role in the metabolism and has a number of functions in the body, including glycogen storage, decomposition of red blood cells, synthesis of plasma proteins, and detoxification. The liver also is the largest gland in the human body. It lies below the diaphragm in the thoracic region of the abdomen. It produces bile, an alkaline compound which aids in digestion, via the emulsification of lipids. It also performs and regulates a wide variety of high-volume biochemical reactions requiring specialized tissues.
Hepatocytes make up 70 to 80% of the cytoplasmic mass of the liver. Hepatocytes are involved in protein synthesis, protein storage and transformation of carbohydrates, synthesis of cholesterol, bile salts and phospholipids, and detoxification, modification and excretion of exogenous and endogenous substances. The hepatocyte also initiates the formation and secretion of bile.
There are a wide number of known liver diseases, such as:                Hepatitis: inflammation of the liver, caused mainly by various viruses but also by certain poisons, autoimmunity or hereditary conditions;        Cirrhosis: the formation of fibrous tissue in the liver, replacing dead liver cells. The death of the liver cells can for example be caused by viral hepatitis, alcoholism or contact with other liver-toxic chemicals;        Haemochromatosis: a hereditary disease causing the accumulation of iron in the body, eventually leading to liver damage;        Cancer of the liver: primary hepatocellular carcinoma (HCC) or cholangiocarcinoma and metastatic cancers, usually from other parts of the gastrointestinal tract;        Wilson's disease: a hereditary disease which causes the body to retain copper;        Primary sclerosing cholangitis: an inflammatory disease of the bile duct, autoimmune in nature;        Primary biliary cirrhosis: autoimmune disease of small bile ducts;        Budd-Chiari syndrome: obstruction of the hepatic vein;        Gilbert's syndrome: a genetic disorder of bilirubin metabolism, found in about 5% of the population;        Glycogen storage disease type II: the build-up of glycogen causes progressive muscle weakness (myopathy) throughout the body and affects various body tissues, particularly in the heart, skeletal muscles, liver and nervous system.        
There are also many pediatric liver diseases, such as biliary atresia, alpha-1-antitrypsin deficiency, alagille syndrome, and progressive familial intrahepatic cholestasis; as well as metabolic diseases.
Furthermore, several pathogens and parasites, especially of tropical diseases, have a liver stage during their life cycle. For instance malaria, is one of the most common infectious diseases and an enormous public-health problem. Malaria is caused by protozoan parasites of the genus Plasmodium. The most serious forms of the disease are caused by Plasmodium falciparum and Plasmodium vivax, but other related species (Plasmodium ovale, Plasmodium malariae, and sometimes Plasmodium knowlesi) can also infect humans.
Another example is Hepatitis B virus (HBV), the cause for hepatitis B. HBV is the prototype of a family of small, enveloped DNA viruses of mammals and birds (11). The HBV envelope encloses three proteins termed L-(large), M-(middle) and S-(small) (see FIG. 1). They share the C-terminal S-domain with four transmembrane regions. The M- and L-protein carry additional N-terminal extensions of 55 and, genotype-dependent, 107 or 118 aa (preS2- and preS1). In virions the stoichiometric ratio of L, M and S is about 1:1:4, while the more abundantly secreted non-infectious subviral particles (SVPs) contain almost exclusively S- and only traces of L-protein (2). During synthesis, the preS1-domain of L is myristoylated and at some point of the HBV life cycle translocated through the ER-derived membrane. This modification is essential for infectivity (13, 14). A pronounced feature of HBV is its liver tropism, i.e. the liver is the tissue which specifically supports the growth of HBV.
Chronic HBV and HCV (Hepatitis C Virus) infections are also major causes for neoplastic alterations in the liver. Primary hepatocellular carcinoma (HCC), usually develop in the setting of chronic liver disease, particular of viral hepatitis. HCC diagnosis can be difficult, usually requiring the use of serum markers and imaging modalities as well as histologic confirmation following biopsy.
HCC results into about one million deaths per annum globally, the median survival following diagnosis is approximately 6 to 20 months (11). One reason for this is the absence of pathogenic symptoms and the liver's large functional reserve (12). As a result the majority of patients diagnosed with hepatocellular carcinoma are not eligible for surgical resection but require other treatment modalities such as liver transplantation, radiofrequency ablation, transarterial chemoembolization, cryoablation or radiation therapy. All of these alternative options are of limited efficacy due to the typically large tumour size at the time of diagnosis. (13) As a consequence the FDA (Federal Drug Agency) recommends intense diagnostic imaging in patients with an underlying liver disease (i.e. cirrhosis, viral hepatitis) who develops a rising serum alpha-fetoprotein level. (12) Diagnostic imaging is usually performed by CT scan of the liver and/or magnetic resonance imaging (MRI). However the initial diagnosis even when using high resolution imaging methods is often difficult as such “follow up” imaging is recommended in the majority of all patients. (14) Novel liver specific contrast agents such as the one described in the invention may help elevate this problem.
Another source for neoplastic alterations in the liver is metastatic spread of primary tumours of other organs, most prominently metastatic spread of colorectal carcinoma (CRC). CRCs are the third most common cause of cancer related death worldwide. About 1.25 million patients develop colorectal cancer per annum; more than 600.000 patients die per year. (Source: GLOBOCAN 2008, globocan.iarc.fr). While the pathogenesis of CRCs is mostly cleared up by now and the FDA recommends regular medical examination for all people from the age of 50 the incidence rate decreased only mildly from 1997 to 2008. (15, 16).
Following primary diagnosis about 80% of all patients do present without any detectable metastasis, for those patients surgical resection is the best therapeutic option. Despite resection more than 40% of all patients diagnosed with TMN classification II/III develop metastasis in the liver (17-21). It has been shown that intensive high resolution imaging following primary resection can significantly enhance the survival time of patients classified in TMN II/III. Early diagnosis of small metastasis in the liver can enhance 5 year survival time following partial hepatectomy by 40% when compared to conventional care. However due to the amorphous structure of the liver early stage diagnosis is often not possible (22). Contrast agents such as the one described herein can help to diagnose liver metastasis in an early stage, allowing surgical intervention and by this help to enhance disease free survival.
Ideally, drug targeting should fulfil the following criteria: 1) exclusive transfer of the drug (e.g. a label) to the required site of action; 2) a minimum of effects for the remaining organism; 3) use of a pharmacologically inactive vector.
In order to carry a label to a specific tissue different strategies are pursued. These are for example the use of prodrugs, from which the pharmacologically active part is released in the target tissue by tissue-specific enzymes. A further possibility is to couple effective, non-tissue-specific drugs to tissue-specific, but pharmacologically inert carrier systems like receptor-affine peptides or colloidal particles.
Various drug carriers have been used to enhance liver targeting of drugs. A straightforward approach is based on the active phagocytosis of the reticuloendothelial system in the liver by delivering drugs in particular carriers, such as liposomes and microspheres. For example, it has been shown that following i.v. (intravenous) injection, particulate carriers incorporating a drug are mainly captured by the reticuloendothelial system in the liver, resulting in drug targeting of the liver (5). On the other hand, liver targeting of drugs with positively charged, water-soluble polymers is based on free extravasation of most water-soluble substances from the vascular system of the liver as well as on negative charges on the liver cell surface (6). Thus, polymers have been used as the carrier to allow drugs to target to the liver based on such anatomical and biochemical characteristics of the liver. More specific drug targeting of the liver has been attempted by using asialoglycoprotein receptors of liver cells. The asialoglycoprotein receptor (galactose receptor) is present on hepatocytes with high density. In addition, once a ligand binds to the galactose receptor, the ligand-receptor complex is internalized which allows the cellular uptake of galactosylated ligands (7). Furthermore delivery approaches using nanoparticles have been performed by e.g. amphilic polymers and viral vectors (8). Also delivery of drugs and genetic material has been conducted via the use of bio-nanocapsules (BNCs). BNCs are described as “nano-scaled capsules consisting of proteins produced by biotechnological techniques” and can be used as delivery systems for organ specific drug delivery (9).
U.S. Pat. No. 7,001,760 B2 disclose recombinant vectors derived from hepatitis B virus (HBV), which can be used for gene therapy, such as the delivery of therapeutic genes to liver cells and the expression of heterologous genes in liver cells.
WO2009/092612, whose content is incorporated herewith by reference in its entirety, describes hydrophobic modified preS-derived peptides of HBV and their use as vehicles for the specific delivery of compounds to the liver. In this document, the hydrophobic modified preS-derived peptides of HBV may be coupled with a diagnostic or therapeutic active agent via an optional anchor group (A) which is preferably “C-terminal” of the hydrophobic modified preS-derived peptide.
The present invention provides hydrophobic modified peptides conjugated to one ore more label which label is coupled to the N-terminal amino acid sequence of the peptide represented by X, making it possible to create shorter peptides while still maintaining liver specificity. Surprisingly it has become possible to couple hydrophobic moieties to the peptides without abrogating liver tropism. Furthermore, coupling labels to the N-terminal site allows better delivery of active compounds across the cell membrane and also allows to direct labels, which are active on the cellular membrane, directly to the site of action. In FIG. 3, the molecular mechanism of the binding of a hydrophobic modified peptide to the surface of a hepatocyte is schematically illustrated.